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Shuttle-Streaming: Synchronization with Heat Production in Slime MoldAuthor(s): Robert D. Allen, W. Reid Pitts, Jr., David Speir, James BraultSource: Science, New Series, Vol. 142, No. 3598 (Dec. 13, 1963), pp. 1485-1487Published by: American Association for the Advancement of ScienceStable URL: http://www.jstor.org/stable/1712013 .

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Shuttle-Streaming: Synchronizationwith Heat Production in Slime Mold

Abstract. Two small blobs and achannel excised from a slime moldplasmodium were allowed to fuse intoa dumbbell-shaped mass in a thermallyinsulated Kamiya double chamberequipped with naked bead thermistorsin contact with the blobs. Cyclic tem-perature differences of from 1 X 10-4to 5 X 10O-deg Celsius were recordedby a sensitive lock-in amplifier methodwith a basal noise level of less than2 X 10-5 deg Celsius and a time con-stant of 0.5 second. The temperaturedifferences were caused by periodicbursts of heat production synchronizedperfectly with the shuttle-streamingcycle and invariably localized at thesource rather than the destination ofthe streaming cytoplasm. The resultssupport the theory that the motive forcefor cytoplasmic streaming in the slimemold is pressure, probably generatedby contraction of elements in the chan-nel walls.

The two central problems in theanalysis of the mechanism of cyto-plasmic streaming phenomena at thecellular or organismal level are (i)identification of the motive force (thatis, as pressure, tension, or other force),and (ii) localization of the site of itsapplication within the cell, or in thecase of the slime mold, the plasmodi-um.

The solutions to these problemsare not so simple as some have be-lieved. Accounts of shuttle-streaming,ameboid movement, cytokinesis, mi-tosis and other dynamic cellular eventsoften include the description of "con-tractions." We claim that the humaneye is capable only of observing de-formations, and has not been demon-strated to have receptors for determin-ing which deformations are active orpassive (1). By way of contrast, mus-cular contractions can be measuredisometrically as tension, or isotonicallyas work performed.The present experiments, designed tolocalize the site of application of themotive force for shuttle-streaming inthe slime mold, are based on the well-known axiom of classical muscle physi-ology, that during contraction sig-nificant amounts of heat are invariablyreleased (2), and on the assumptionthat the same principle should holdtrue for the mechanochemical processes13 DECEMBER 1963

responsible for cytoplasmic streamingin the slime mold.Plasmodia of Physarum polycepha-lum were cultured on filter papersaturated with tap water and sprinkledwith oatmeal (3), and then allowed tofast for a day before two blobs (about

5 mm in diameter) and a channel(about 6 mm long, 0.2 mm in diam-eter) were excised and allowed to fuseinto a dumbbell-shaped mass in amodified Kamiya double chamber(4). The chambers were made of lu-cite (Fig. 1) and so designed thatsmall bead thermistors (5) (0.1 mm indiameter) would lie in contact withthe terminal blobs. The connectingchannel was embedded in vaseline anda cover glass placed over the twocompartments. The double chamberwas then lowered into a thermally in-sulated (styrofoam) box fitted withdouble-layer plastic windows adequatefor observing the direction of streamingwith a dissecting microscope.Leads from the thermistors werepassed through the insulated box to op-posite sides of a Wheatstone bridgecircuit containing two additional re-sistances, one of which was a sensitivepotentiometer for balancing the bridge(Fig. 2). An oscillator in the lock-inamplifier (6) provided a 400 cy/secmodulation for the bridge. Any thermalimbalance between the two thermistorsresulted in a signal at the single-endedoutput across the bridge. This signalwas amplified by a vacuum-tube volt-meter (7) before reaching the tunedamplifier of the lock-in. The use ofa lock-in amplifier allowed two majorimprovements over a somewhat similarthermistor thermometer described byStow (8) in that the phase-sensitivedetector allowed the sign of the temper-ature differences to be registered, andthe narrow-band detection afforded bythis technique resulted in a significantreduction in the basal noise level (9).The deflection sensitivity was deter-mined by direct calibration in solutionsof known temperatures a few degreesapart with the instrument operated atvery low gains. The basal noise levelwas determined by allowing wads ofmoist cotton to lie in contact with thethermistors until thermal equilibriumhad been established. At maximumgain, the rms noise was equivalent toless than 2 X 10-5 deg Celsius whenthe filtering time constant of the lock-in amplifier was matched with that ofthe thermistors ( - 0.5 second).

p

Fig. 1. A diagram of the modifiedKamiya double chamber showing the po-sition of the thermistors (T), pressure out-lets (P) and terminals connected to thethermistor leads (L).

The measurements reported in thepresent study were comparable in sensi-tivity to the elegant thermopile-galvano-meter measurements of heat productionin muscle and nerve by A. V. Hill andco-workers (10). The present tech-nique was chosen principally for itssimplicity, convenience and economy.In addition, thermistors are availablecommercially in sizes considerablysmaller than the plasmodial blobs.A 30- to 50-minute interval of ther-mal isolation was required to ensurethe onset of vigorous cytoplasmicstreaming and to allow thermal pertur-bations in the double chamber to sub-side. To visualize streaming, a mono-chromatic light source was chosen tolie outside the normal absorption rangeof the plasmodial pigments (220 to440 mt) (11). The mercury greenline (546 mt) of a General ElectricAH-4 lamp was filtered by 2 cm ofwater, an interference filter, and a vari-able-density polarizing filter adjusted totransmit barely enough light to renderthe direction of streaming visible.The tendency of plasmodia to mi-grate caused some to lose contact withone or both thermistors, and therefore

LOCK-IN AMPLIFIER

i II- IVNED i(plEsu lle PlltUNF D . PozsE- tD iTP-. 1CrsI AUPL.I.lR t f ..... . SH*tn T os;1,1ATI I

Fig. 2. A block diagram of the differentialthermometer.1485

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only about half of the preparationsshowed thermal cycles. In the remain-ing 20 successful experiments, the tem-perature differences recorded rangedfrom 1 X 10-4 and 5 X 10-' deg.Celsius. The wide differences in magni-tude probably reflected not only themovement of plasmodia out of con-tact with the thermistors, but a tend-ency of some plasmodia to secrete aninsulating layer of slime.

Changes in the sign of the tempera-ture difference were perfectly syn-chronized with changes in the direc-tion of streaming. Invariably, thegreater heat production was detectedat the source of the streaming cyto-plasm (Fig. 3). It was established thatthe thermal cycles were produced bythe plasmodia and not by instrumentalfactors by the following facts. (i) Insome experiments which were initiatedin the dark, there were thermal cyclesbefore the illumination was turned on;the light neither caused nor detectiblyaffected the thermal cycles. (ii) In con-trol experiments with wads of moistcotton, there were no thermal cycles.(iii) The possibility that the thermalcycles were produced by the transportaway from the thermistors of cyto-plasm heated by the thermistors waseliminated by measuring the thermalcycles in the same plasmodium ata constant deflection sensitivity main-tained by various different combina-tions of input voltage to the bridge andamplifier gain. No correspondence wasfound between the input voltage tothe bridge and the amplitude of thethermal cycles found in the plasmo-dium.

A single blob of plasmodium meas-ured against a wad of moist cottonshowed a gradual tendency to becomewarmer relative to the cotton coupledwith nonrhythmic bursts of heat pro-duction (Fig. 4). Two blobs of plas-modium not connected by a strandshowed similar but smaller bursts ofthermal activity without rhythmicity(Fig. 5). Similarly, dumbbell-shapedplasmodia which had shown thermalcycles lost their rhythmicity when theconnecting channel was excised. Oneexception was an experiment in whichthere remained a passage of water inthe vaseline seal between the two blobs.The observation of thermal cycles inthis experiment led us to connect twoplasmodial blobs with a wet cottonstring, a procedure found by Kamiyaand Abe (12) to permit the continuanceof surface potential difference waves.Under these conditions, thermal cycles

1486

of the characteristifound (Fig. 6) allnot so regular asdumbbell-shaped plFigs. 3 and 6).

Opinions have diflshuttle-streaming inshould be considere

AT(x 1o 5e)

400.

350 0

300

250

200

150

100-L

I x?'~i !v I0 2

Fig. 3. A sample ofperature differencesin contact with the t(see Fig. 1).

R,M.S. NOISE LE

( x1 5 ) 50

0 2 4 6

Fig. 4. A record offerences between ablob and a piece ofroughly equal size.

250 R.MS,.NOISE LEV

200 -AT

(Xo 's5) 15010050

2 4 6

Fig. 5. A record offerences between twthermally and electrione another.

450400350300

( x 10to5) 200150t0050

Fig. 6. A record offerences between twconnected only by a

ic frequency were phenomenologically distinct from otherthough they were examples of cytoplasmic streamingthose observed in (such as ameboid movement, and[asmodia (compare cytoplasmic rotation), or to be onemanifestation of a universal (but un-fered as to whether known) mechanism of cytoplasmicthe slime mold streaming. Seifriz, on observing time-:d to be a process lapse motion pictures of the "pulsa-tions" in plasmodia (13), advanced thetheory that streaming in slime moldsL was induced by pressure differencesresulting from localized contractions.

LLater, he abandoned this theory on theL l I v i basis that it was incompatible with thedetails of streaming phenomena inA /v I other organisms (14). Hilton (15) wasL I [/V probably the first to show that thedirection of streaming could be shiftedby mechanical pressure applied to the

t R plasmodium from outside, and heconcluded that streaming was caused by"expansions and contractions" of the.MINUTESE plasmodial walls. More recently, Kami-ya and Kuroda (16) showed that thea record of the tem-ba eco therstors truncated velocity profiles across abetween thermistorswo plasmodial blobs main channel were the same, whetherthe streaming was natural or induced

by gas pressure; on this basis it wasreasoned that pressure was the normalEVEL .3 X 10'5 motive force, although Kamiya (17,

18) later admitted that (i) "suction"^ . .......ornother force acting from the front,

MINUTES or (ii) active shearing along thinnerchannel walls, could not be eliminated asthe temperature dif- an explanation. The latter (active shear-single plasmodialmoistened cotton of ing) was said possibly to offer a com-mon mechanism for shuttle-streamingand rotational streaming. The truncatedvelocity profiles would also be com-EL: 2.3 X1-' patible with a frontal contraction mech-

anism, such as that recently proposedfor the ameba by Allen (19), parti-cularly in view of some of the com-plexities of movement that have been

.------ pointed out by Kamiya (17) and by thea 10 12 14 16MINUTES Stewarts (20, 21). That is, it is con-ceivable that contractions at the frontthe temperature dif-o plasmodial blobis (destination) of cytoplasmic streamscally insulated from might exert tension on structural ele-ments which have now been demon-strated to exist in the axial region ofthe endoplasmic stream of the slime

mold (16, 21).The present results appear to estab-4OSELEVEL 2.3 X 105 lish that if streamingcan be attributedto a single motive force mechanism,that force must be applied where the

greater part of the heat synchronizedwith the streaming cycle is produced;\/\ that is, at the origin of the stream. If-- - more than one motive force is in op-8 I0 12 14 16 18

MINUTES eration, they must either operate in thete t e d- same general region of the plasmo-the temperature dif-vo plasmodial blobs dium, or the second force not appliedwet cotton thread. at the source must be much less im-

SCIENCE, VOL. 142

5 0 -

R.M.S. N

4 6

A 11-1 _ _-

LI

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portant quantitatively than the mainmotive force responsible for the ther-mal cycles which we have found.While the possibility, admitted byKamiya (17), that the motive forcemight be due to interactions betweenthe smaller streams and their channelwalls cannot be rigorously excludedon the basis of all the information nowavailable, there is increasing evidenceto suggest that the mechanochemicalevent responsible for streaming is acontraction of the channel wall mater-ial. For example: (i) the occurrence ofpulsations, which may be interpretedas contractions; (ii) the ability ofhanging plasmodial strands to showboth torsional motions and isotoniccontractions (23); (iii) microscopi-cally observable changes in channeldiameter during the streaming cycle;(iv) the demonstration of a proteinsystem capable of both mechanochemi-cal and enzymatic reactions similar tothose of muscular "contractile proteins";and finally (v), the recent demonstra-tion of a dynamically organized fibril-lar system the form and birefringenceof which fluctuates during the streamingcycle (24).We would prefer not to generalizefrom our present evidence concerningthe mechanism of slime mold stream-ing to problems in the mechanism ofameboid movement, reticulopodialstreaming in foraminifera, and rota-tional streaming in Characeae, whererecent studies suggest diversity of mech-anism at the cellular level (24). Thisdoes not exclude the possibility thatthe molecular basis of cytoplasmicstreaming phenomena might show theunity of mechanism which many haveexpected to find at the cellular level(25). ROBERTD. ALLEN

W. REID PITTS, JR.DAVID SPEIR

Department of BiologyJAMES BRAULT

Department of Physics, PrincetonUniversity, Princeton, New Jersey

References and Notes1. The direct observation of cytoplasmic con-tractions is possible only by "inferencemicroscopy."2. A summary of the work of A. V. Hill andco-workers on the thermal aspects of musclecontraction is given by H. Davson, in A Text-book of General Physiology (Little, Brown,Boston, ed. 2, 1959), pp. 662-686.3. W. G. Camp, Bull. Torrey Botan. Club 63,205 (1936).

portant quantitatively than the mainmotive force responsible for the ther-mal cycles which we have found.While the possibility, admitted byKamiya (17), that the motive forcemight be due to interactions betweenthe smaller streams and their channelwalls cannot be rigorously excludedon the basis of all the information nowavailable, there is increasing evidenceto suggest that the mechanochemicalevent responsible for streaming is acontraction of the channel wall mater-ial. For example: (i) the occurrence ofpulsations, which may be interpretedas contractions; (ii) the ability ofhanging plasmodial strands to showboth torsional motions and isotoniccontractions (23); (iii) microscopi-cally observable changes in channeldiameter during the streaming cycle;(iv) the demonstration of a proteinsystem capable of both mechanochemi-cal and enzymatic reactions similar tothose of muscular "contractile proteins";and finally (v), the recent demonstra-tion of a dynamically organized fibril-lar system the form and birefringenceof which fluctuates during the streamingcycle (24).We would prefer not to generalizefrom our present evidence concerningthe mechanism of slime mold stream-ing to problems in the mechanism ofameboid movement, reticulopodialstreaming in foraminifera, and rota-tional streaming in Characeae, whererecent studies suggest diversity of mech-anism at the cellular level (24). Thisdoes not exclude the possibility thatthe molecular basis of cytoplasmicstreaming phenomena might show theunity of mechanism which many haveexpected to find at the cellular level(25). ROBERTD. ALLEN

W. REID PITTS, JR.DAVID SPEIR

Department of BiologyJAMES BRAULT

Department of Physics, PrincetonUniversity, Princeton, New Jersey

References and Notes1. The direct observation of cytoplasmic con-tractions is possible only by "inferencemicroscopy."2. A summary of the work of A. V. Hill andco-workers on the thermal aspects of musclecontraction is given by H. Davson, in A Text-book of General Physiology (Little, Brown,Boston, ed. 2, 1959), pp. 662-686.3. W. G. Camp, Bull. Torrey Botan. Club 63,205 (1936).4. N. Kamiya, Science 92, 2394 (1940).5. Manufactured by the Victory EngineeringCo., Springfield, N.J. Catalog No. 32A130.6. Model JB-4 (or JB-5) lock-in amplifier,manufactured by the Princeton Applied Re-search Corporation, Princeton, N.J.7. Model 400 D vacuum tube voltmeter, manu-

13 DECEMBER 1963

4. N. Kamiya, Science 92, 2394 (1940).5. Manufactured by the Victory EngineeringCo., Springfield, N.J. Catalog No. 32A130.6. Model JB-4 (or JB-5) lock-in amplifier,manufactured by the Princeton Applied Re-search Corporation, Princeton, N.J.7. Model 400 D vacuum tube voltmeter, manu-13 DECEMBER 1963

factured by the Hewlett Packard Company,Palo Alto, Calif.8. R. W. Stow, Rev. Sci. Instr. 29, 774 (1958).9. A similar technique has been described formeasuring small phase retardations of el-liptically polarized light; see R. D. Allen,J. Brault, R. D. Moore, J. Cell Biol. 18, 223(1963).10. A. V. Hill, Proc. Roy. Soc. London, Ser. B.124, 114 (1937); see also (2).11. F. T. Wolf, in Photoperiodism and RelatedPhenomena in Plants and Animals, AAASPubl. No. 55, R. B. Withrow, Ed. (Wash-ington, D.C., 1959), p. 321.12. N. Kamiya, and S. Ab6. J. Colloid Sci. 5,149 (1950).13. W. Seifriz, Science 86, 2235 (1937).14. - , Nature 171, 1136 (1953).15. A. E. Hilton, J. Quekett Microscop. Club10, 263 (1908).16. N. Kamiya and K. Kuroda, Protoplasma44, 1 (1958).17. N. Kamiya, "Protoplasmic Streaming," Proto-plasmatologia, No. 8 (1959), pp. 142-144,169-171.18. N. Kamiya, Ann. Rep. Sci. Works, Fac. Sci.Osaka Univ. 8, 13 (1960), see p. 39.19. R. D. Allen, Exptl. Cell Res. Suppl. 8, 17(1961).20. P. A. Stewart, and B. T. Stewart, Exptl. CellRes. 17, 44 (1959).21. P. A. Stewart, in Primitive Motile Systems in

factured by the Hewlett Packard Company,Palo Alto, Calif.8. R. W. Stow, Rev. Sci. Instr. 29, 774 (1958).9. A similar technique has been described formeasuring small phase retardations of el-liptically polarized light; see R. D. Allen,J. Brault, R. D. Moore, J. Cell Biol. 18, 223(1963).10. A. V. Hill, Proc. Roy. Soc. London, Ser. B.124, 114 (1937); see also (2).11. F. T. Wolf, in Photoperiodism and RelatedPhenomena in Plants and Animals, AAASPubl. No. 55, R. B. Withrow, Ed. (Wash-ington, D.C., 1959), p. 321.12. N. Kamiya, and S. Ab6. J. Colloid Sci. 5,149 (1950).13. W. Seifriz, Science 86, 2235 (1937).14. - , Nature 171, 1136 (1953).15. A. E. Hilton, J. Quekett Microscop. Club10, 263 (1908).16. N. Kamiya and K. Kuroda, Protoplasma44, 1 (1958).17. N. Kamiya, "Protoplasmic Streaming," Proto-plasmatologia, No. 8 (1959), pp. 142-144,169-171.18. N. Kamiya, Ann. Rep. Sci. Works, Fac. Sci.Osaka Univ. 8, 13 (1960), see p. 39.19. R. D. Allen, Exptl. Cell Res. Suppl. 8, 17(1961).20. P. A. Stewart, and B. T. Stewart, Exptl. CellRes. 17, 44 (1959).21. P. A. Stewart, in Primitive Motile Systems in

Since Dmochowski and Grey (1) in1957 observed virus-like particles inthin sections of AK mice with spon-taneous leukemia, and in C3H miceinjected with Gross leukemia virus atbirth, the thin-section technique hasbeen widely used in attempts to demon-strate similar virus-like particles in theleukemic cells and tissues of man.Dmochowski (2, 3) has recently re-viewed his and others' findings in thisfield. As Bryan (4) concludes, virus-like particles have been reported inonly a small fraction of leukemicpatients examined.The negative staining technique (5)for the study of virus particles withthe electron microscope permits thefine structure of virus particles to bediscerned in great detail, making iteasier to decide whether any givenparticle resembles a known virus par-ticle, and also permits virus particlesto be classified on a morphologicalbasis (6, 7). By a modification of thetechnique recently evolved in ourlaboratory (8, 9) virus particles can benegatively stained in what are essen-tially untreated cell preparations. Thismethod does not require purificationprocedures which might destroy afragile virus particle. Hence even the

Since Dmochowski and Grey (1) in1957 observed virus-like particles inthin sections of AK mice with spon-taneous leukemia, and in C3H miceinjected with Gross leukemia virus atbirth, the thin-section technique hasbeen widely used in attempts to demon-strate similar virus-like particles in theleukemic cells and tissues of man.Dmochowski (2, 3) has recently re-viewed his and others' findings in thisfield. As Bryan (4) concludes, virus-like particles have been reported inonly a small fraction of leukemicpatients examined.The negative staining technique (5)for the study of virus particles withthe electron microscope permits thefine structure of virus particles to bediscerned in great detail, making iteasier to decide whether any givenparticle resembles a known virus par-ticle, and also permits virus particlesto be classified on a morphologicalbasis (6, 7). By a modification of thetechnique recently evolved in ourlaboratory (8, 9) virus particles can benegatively stained in what are essen-tially untreated cell preparations. Thismethod does not require purificationprocedures which might destroy afragile virus particle. Hence even the

Cell Biology, R. D. Allen and N. Kamiya,Eds. (Academic Press, New York, in press).22. R. D. Allen, S. Cox, J. D. Belcher, paperpresented at the Symposium on Biorheology,Providence, R.I., 1963.23. N. Kamiya and W. Seifriz, Exptl. Cell Res.6, 1 (1954); also unpublished films shownin the U.S. by N. Kamiya during 1962 and1963.24. At a recent symposium, H. Nakajima andK. Wohlfarth-Bottermann presented reportsof polarized light and electron microscopicstudies, respectively, of the fibrillar organi-zation of myxomycete plasmodia. These re-ports will appear in Primitive Motile Sys-terns in Cell Biology, R. D. Allen and N.Kamiya, Eds. (Academic Press, New York,in press). Other papers in this volume willsummarize the state of knowledge regardingvarious protoplasmic streaming systems.Taken together, these accounts seem to ad-mit greater diversity of mechanism at thecellular level than might have been expecteda few years ago.25. Supported by research grant RG-8691from the National Institutes of Health. Wethank Noburo Kamiya and H. Nakajima fortheir helpful suggestions, and William Mad-dux for his participation in the early stagesof this work.18 October 1963 8

Cell Biology, R. D. Allen and N. Kamiya,Eds. (Academic Press, New York, in press).22. R. D. Allen, S. Cox, J. D. Belcher, paperpresented at the Symposium on Biorheology,Providence, R.I., 1963.23. N. Kamiya and W. Seifriz, Exptl. Cell Res.6, 1 (1954); also unpublished films shownin the U.S. by N. Kamiya during 1962 and1963.24. At a recent symposium, H. Nakajima andK. Wohlfarth-Bottermann presented reportsof polarized light and electron microscopicstudies, respectively, of the fibrillar organi-zation of myxomycete plasmodia. These re-ports will appear in Primitive Motile Sys-terns in Cell Biology, R. D. Allen and N.Kamiya, Eds. (Academic Press, New York,in press). Other papers in this volume willsummarize the state of knowledge regardingvarious protoplasmic streaming systems.Taken together, these accounts seem to ad-mit greater diversity of mechanism at thecellular level than might have been expecteda few years ago.25. Supported by research grant RG-8691from the National Institutes of Health. Wethank Noburo Kamiya and H. Nakajima fortheir helpful suggestions, and William Mad-dux for his participation in the early stagesof this work.18 October 1963 8

fine structure of rabies virus particles(which are notoriously fragile and un-stable) has been observed in cell prep-arations (10). For this reason thistechnique has been used to examinethe leukocytes in the blood of patientswith acute leukemia.

Blood from six patients with acuteblast-cell leukemia and blood from sixhealthy subjects have now been studied.Hematological and other clinical find-ings indicated that three of the leu-kemias were lymphoblastic and theother three myeloblastic. In two of themyeloblastic cases, which clinicallywere similar, virus-like particles wereconsistently found in leukocyte prepara-tions, and the particles in each in-stance were of the same morphologicaltype.The first patient was a 43-year oldfemale with symptoms of 2 months'duration. On admission to the hospitalher leukocyte count was 140,000/mm'with 88 percent primitive cells, herhemoglobin was 6.8 g per 100 ml ofblood, and her platelet count 25,000/mm3. The sternal marrow showed 95percent primitive cells. Two specimensof peripheral blood were obtained be-fore, and one 8 days after, the startof 6-mercaptopurine therapy. The sec-

1487

fine structure of rabies virus particles(which are notoriously fragile and un-stable) has been observed in cell prep-arations (10). For this reason thistechnique has been used to examinethe leukocytes in the blood of patientswith acute leukemia.

Blood from six patients with acuteblast-cell leukemia and blood from sixhealthy subjects have now been studied.Hematological and other clinical find-ings indicated that three of the leu-kemias were lymphoblastic and theother three myeloblastic. In two of themyeloblastic cases, which clinicallywere similar, virus-like particles wereconsistently found in leukocyte prepara-tions, and the particles in each in-stance were of the same morphologicaltype.The first patient was a 43-year oldfemale with symptoms of 2 months'duration. On admission to the hospitalher leukocyte count was 140,000/mm'with 88 percent primitive cells, herhemoglobin was 6.8 g per 100 ml ofblood, and her platelet count 25,000/mm3. The sternal marrow showed 95percent primitive cells. Two specimensof peripheral blood were obtained be-fore, and one 8 days after, the startof 6-mercaptopurine therapy. The sec-

1487

Virus-Like Particles in Blood of Two Acute Leukemia PatientsAbstract. A modified negative staining technique suited to demonstratingfragile virus particles in the electron microscope revealed particles having a

surrounding membranous sac and an internal filamentous component of a con-sistent diameter of 75 A in the blood of two patients with acute leukemia. Noparticles of this type were seen in the blood of four other patients with acuteleukemia or in the blood of six normal individuals.

Virus-Like Particles in Blood of Two Acute Leukemia PatientsAbstract. A modified negative staining technique suited to demonstratingfragile virus particles in the electron microscope revealed particles having a

surrounding membranous sac and an internal filamentous component of a con-sistent diameter of 75 A in the blood of two patients with acute leukemia. Noparticles of this type were seen in the blood of four other patients with acuteleukemia or in the blood of six normal individuals.